Aryl hydrocarbon receptor sulfenylation promotes glycogenolysis and rescues cancer chemoresistance

Elevation of reactive oxygen species (ROS) levels is a general consequence of tumor cells’ response to treatment and may cause tumor cell death. Mechanisms by which tumor cells clear fatal ROS, thereby rescuing redox balance and entering a chemoresistant state, remain unclear. Here, we show that cysteine sulfenylation by ROS confers on aryl hydrocarbon receptor (AHR) the ability to dissociate from the heat shock protein 90 complex but to bind to the PPP1R3 family member PPP1R3C of the glycogen complex in drug-treated tumor cells, thus activating glycogen phosphorylase to initiate glycogenolysis and the subsequent pentose phosphate pathway, leading to NADPH production for ROS clearance and chemoresistance formation. We found that basic ROS levels were higher in chemoresistant cells than in chemosensitive cells, guaranteeing the rapid induction of AHR sulfenylation for the clearance of excess ROS. These findings reveal that AHR can act as an ROS sensor to mediate chemoresistance, thus providing a potential strategy to reverse chemoresistance in patients with cancer.

C-glucose were pre-treated with SR1 (10 μM) for 12 hr, and switched to 12 Cglucose for 4 hr drug-treatment, and 13 C-labeled R5P or S7P was detected by LC-MS/MS.(E) DRCs pre-treated with SR1 for 12 hr were treated with DDP or 5-Fu for 24 hr.NADPH/NADP + and ROS levels were analyzed.(F) DRCs cultured in 13 Cglucose medium for 10 days pre-treated with SR1 for 24 hr were treated with DDP or 5-Fu for 8 hr, followed by the treatment with hydrochloric acid, leading to the degradation of polymer glycogen into monomer glucose.The released 13 C-labeled glucose was determined by LC-MS.All error bars are mean ± SD, p values were calculated by one-way ANOVA followed by Bonferroni's test (C), two-tailed unpaired Student's t test (D-F), n=3, **p < 0.01, ***p < 0.001.

Supplementary Tables 1-4
Breast and lung cancer tissues were obtained by needle biopsy before chemotherapy, whereas after chemotherapy, tumor tissues were obtained by surgery.The chemotherapy responders and non-responders were evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST).

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Fig. S1 N-W: DRCs use higher ROS to produce NADPH during treatment.Related to Fig. 1. (N) HyPerRed-expressing DRCs pre-treated with NAC (5 mM) for 12hr were treated with DDP for 24 hr.MFI was analyzed.Scale bar, 10 μm.(O) DRCs pre-treated with NAC (5 mM) for 12 hr were treated with drugs for 48hr.The cell viability was analyzed.(P) DRCs transduced with si-NC or si-G6PD was treated with DDP for 24 hr.GSH/GSSG was analyzed.(Q) DRCs transduced with si-NC or si-GPX1 was treated with DDP for indicate times.ROS level (24 hr) and cell viability (48 hr) were analyzed.(R) DRCs was treated with DDP alone or in combination with GSH synthesis inhibitor (BSO, 50 μM) for indicate times.ROS level and cell viability were analyzed.(S) DRCs was treated with DDP alone or in combination with SOD1 inhibitor (LCS-1, 5 μM) for indicate times.ROS level and cell viability were analyzed.(T) DRCs was treated with DDP alone or in combination with CAT inhibitor (AMT, 50 μM) for indicate times.ROS level and cell viability were analyzed.(U) DSCs and DRCs were treated with DDP

Fig. S2 :
Fig. S2: Glycogenolysis drives PPP in DRCs in response to drug molecules.Related to Fig. 2. (A) DRCs were cultured in 13 C-glucose for 10 days, followed by the treatment with hydrochloric acid, leading to the degradation of polymer glycogen into monomer glucose.The released 13 C-labeled glucose was determined by LC-MS/MS.(B) DRCs were treated with drugs alone or in combination with GPI (50 μM) for 48 hr.The cell

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Fig. S3 A-F: DRCs use AHR to promote glycogenolysis.Related to Fig. 3. (A) A549 or A549/5-Fu cells were treated with 5-Fu for 24 hr.Cell lysates were immunoprecipitated with anti-STBD1 for mass spectrometry.Identified proteins were listed.(B) DRCs were pre-treated with SR1 (10 μM) for 12 hr, and treated with DDP for 24 hr, Phospho-PYGL or PYGL were analyzed by Western Blot.(C) DRCs cultured in 13 C-glucose transduced with si-NC or si-AHR were switched to 12 C-glucose for 4 hr drug-treatment, and 13 C-labeled S7P was detected by LC-MS/MS.(D) DRCs cultured in 13 C-glucose were pre-treated with SR1 (10 μM) for 12 hr, and switched to 12 C-

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Fig. S3 G-O: DRCs use AHR to promote glycogenolysis.Related to Fig. 3. (G-I) The expression of ALDH (G), CD133(H) and CD90 (I) in DSCs (MCF-7, A549) and DRCs (MCF-7/DDP, A549/5-Fu) were determined by flow cytometry.(J-L) The expression of OCT4 (J), SOX2 (K) and β-catenin (L) in DSCs and DRCs were determined by Real-time PCR.(M) The expression of E-Cadherin in DSCs and DRCs were determined by flow cytometry.(N and O) The expression of Vimentin (N) and SNAIL (O) in DSCs and DRCs were determined by Real-time PCR.G-O, n=3.All error bars are mean ± SD, p values were calculated by two-tailed unpaired Student's t test, NS, not significant (p > 0.05).

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Fig. S5 K-O: Sulfenylated AHR promotes GP activity by competitively binding PTG.Related to Fig. 5. (K) Co-IP of S-glutathionylation of AHR in DRC (MCF-7/DDP) (IP, GSH; IB, AHR).(L) Immunoblot of immunoprecipitations of PTG in lysates from DRCs or GRX1-over expressing DRCs treated with DDP for 24 hr.(M) Immunoblot of immunoprecipitations of p-PYGL or PTG in lysates from DRC (MCF-7/DDP) transfected with si-NC or si-GRX1 treated with DDP for 24 hr.(N and O) The G6PD activity (N) and the ratio of NADPH/NADP+ (O) in DRCs transfected with si-NC or si-GRX1 was analyzed (n=3).All error bars are mean ± SD, p values were calculated by one-way ANOVA followed by Bonferroni's test (N, O), n=3, NS, not significant (p > 0.05).

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Fig. S6 A-P: Sulfenylated AHR-regulated glycogenolysis promotes drug resistance in vivo.Related to Fig. 6. (A and B) Tumor-bearing mice were administrated with 5-Fu.Tumor growth (A) and mouse survival (B) were monitored.(C-E) Tumor-bearing mice were administrated with drugs for 5 times.ROS levels and NADPH/NADP + in isolated tumor cells were analyzed (C), and R5P, S7P or E4P levels were determined

Fig. S7 :
Fig. S7: Sulfenylated AHR-regulated glycogenolysis promotes drug resistance in vivo.Related to Fig. 6. (A and B) Tumor-bearing mice were administrated with drugs or SR1 for 3 times.p-PYGL and PYGL expression were determined (A), and ROS levels or NADPH/NADP + (B) in isolated tumor cells were analyzed.(C) shScr, shAHR 1 or shAHR 2 were inoculated into NSG mice administrated with drugs for 3 times.ROS levels and NADPH/ NADP+ in isolated tumor cells were analyzed.(D and E) Tumor-bearing mice were administrated with drugs or SR1.The tumor growth and the survival of mice were monitored.(F) shScr, sh-AHR 1 or shAHR 2 DRCs were

Fig. S8 :
Fig. S8: AHR-glycogenolysis occurs in chemo-resistant cancer patients.Related to Fig. 7. (A-C) Overall survival compared to the AHR level in people with glioma (n = 529), and pancreatic adenocarcinoma (n = 182) (A), and PYGL level in people with colon cancer (n = 698) (B), and G6PD level in people with colon cancer (n = 698) (C).(D and E) The tissue sections from lung cancer patients including responders or non-